Fig. 5.

Catechin suppression of NNK- and B[a]P-induced short-term-targeted end points in MCF7 cells. MCF7 cells were exposed to combined NNK and B[a]P (NB) each at 100 pmol/l in the absence or presence of 10 μg/ml of EC, ECG, EGC and EGCG for 24 h. (A) ROS levels were measured with chloromethyl-dichlorodihydro-fluorescein-diacetate labeling; relative level of ROS as fold induction was normalized by the level determined in untreated counterpart cells, set as 1 (X, arbitrary unit). (B and D) Cell lysates were prepared and analyzed by western immunoblotting to detect levels of phosphorylated Erk1/2 (p-Erk1/2), Erk1/2, p-H2AX and H2AX, with β-actin as a control, and these levels were quantified by densitometry. The levels of specific phosphorylation Erk1/2 (p-Erk) and H2AX (p-H2AX) were calculated by normalizing the levels of p-Erk1/2 and p-H2AX with the levels of Erk1/2 and H2AX, respectively, and then further normalizing with β-actin level and the level set in untreated cells (lane 1) as 1 (X, arbitrary unit). (C) Cell proliferation was determined; relative cell growth rate was normalized by the value of BrdU detected in untreated counterpart cells, set as 1 (X, arbitrary unit). Columns, mean of triplicates; bars, SD. All results are representative of at least three independent experiments. The Student’s t-test was used to analyze statistical significance, indicated by **P < 0.01, ***P < 0.001; α levels were adjusted by the Simes method.